Azide based reagents are used extensively by NIH supported investigators in photoaffinity labelling and cross-linking studies. Most practitioners utilize a few commercially available azide reagents. It is generally believed that the photochemistry of these reagents in water is similar to that of simple phenyl azides in organic solvents. Preliminary results indicate that this assumption is not generally correct. Thus, we will systematically study the photochemistry of widely used commercially available reagents, simple phenyl azides, cinnamyl, sulfonyl, phoshoryl and acyl azides in water and in buffer solution. We will study these reagents by pico, nano and microsecond time-resolved UV-Vis and IR spectroscopy, which will reveal the lifetimes of the light generated reactive intermediates and absolute reactivities of the intermediates with nucleosides and amino acids. The reactive intermediates will be identified with the aid of CASSCF, CASPT2 and DFT calculations and chemical analysis of reaction mixtures. This work will be done with non-NIH resources. With NIH support a series of new azide precursors will be synthesized, which absorb strongly above 330 nm and reliably produce reactive intermediates in water with lifetimes of 0.1-100 ns. In collaboration with OSU colleague Professor Ming-Daw Tsai we will re-investigate a classic system, the E. coli Klenow Fragment I which has been previously studied with a traditional, commercial reagent (8-azido dATP) and a newly developed reagent to demonstrate that the commercial reagent generates untrustworthy results. In collaboration with a second OSU colleague, Professor Mike Freitas, we will study the labelled Klenow Fragment with high throughput Mass Spectrometric techniques. These new reagents will also be evaluated as cross-linking agents by Professors Pei and Dalbey of The Ohio State University (OSU) to cross-link the PNAG and YidG proteins, respectively. A photo-probe group will be created at OSU bringing together synthetic organic chemists, physical-organic chemists, mass spectrometrists and biochemists.